(1) The mechanism of activation of calmodulin-dependent phosphoprotein phosphatase by a crucial divalent metal ion like Ni2+ or Mn2+ has been studied under diverse conditions. Activation of the phosphatase by Ni2+ is characterized by an initial lag period and by extremely tight binding. Different levels of Ni2+ give rise to the same final extent of activity, although the lag time decreases as Ni2+ concentration is raised. In the presence of calmodulin, only one Ni2+ ion is bound, the lag phase conforms with first-order kinetics, and the initial enzyme Ni2+ complex is inactive. The initial dissociation constant for Ni2+ is 2.5 mM and the rate constant for the activation process (conformational rearrangement) is 0.083s minus 1. In the absence of calmodulin, the lag phase no longer conforms with a first-order process. Both kinetic and binding experiments indicate the involvement of two Ni2+ ions. It appears that the additional Ni2+ binding site overlaps with the calmodulin binding domain on the phosphatase. Activation by Mn2+ in the presence of calmodulin is similar to that by Ni2+ except that the process is reversible. The initial enzyme Mn2+ binding constant, 200 Mum, is tighter, but the activation rate constant, 0.042s minus 1, is somewhat slower compared with corresponding parameters for Ni2+. (2) The activation of calmodulin-regulated enzymes by Ca2+ was treated theoretically by assuming all the components involved were in rapid equilibria. But the extremely slow off-rate of calmodulin from the enzyme-calmodulin complex seems to contradict the assumption. A mathematical derivation has been developed which demonstrates that the rapid-equilibrium assumption is valid as long as the binding of Ca2+ to various enzyme calmodulin complexes are rapid. (3) The calmodulin-dependent phosphoprotein phosphatase was found to be phosphorylated by the Ca2+-dependent C kinase. The phosphorylated form of calmoldulin-dependent protein kinase in dephosphorylated by the calmodulin-dependent phosphatase.